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. 2018 Sep 28;13(9):e0204271.
doi: 10.1371/journal.pone.0204271. eCollection 2018.

The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro

Xuan Wang  1 , Andris Elksnis  1 , Per Wikström  2 , Erik Walum  2 , Nils Welsh  1 , Per-Ola Carlsson  1
Affiliations

The novel NADPH oxidase 4 selective inhibitor GLX7013114 counteracts human islet cell death in vitro

Xuan Wang et al. PLoS One. .

Abstract

It has been proposed that pancreatic beta-cell dysfunction in type 2 diabetes is promoted by oxidative stress caused by NADPH oxidase (Nox) over-activity. The aim of the present study was to evaluate the efficacy of novel Nox inhibitors as protective agents against cytokine- or high glucose + palmitate-induced human beta-cell death. The Nox2 protein was present mainly in the cytoplasm and was induced by cytokines. Nox4 protein immunoreactivity, with some nuclear accumulation, was observed in human islet cells, and was not affected by islet culture in the presence of cytokines or high glucose + palmitate. Nox inhibitors with partial or no isoform selectivity (DPI, dapsone, GLX351322, and GLX481372) all reduced ROS production of human islet cells exposed to high glucose + palmitate. This was paralleled by improved viability and reduced caspase 3 activation. The Nox1 selective inhibitor ML171 failed to reduce human islet cell death in response to both cytokines and high glucose + palmitate. The selective Nox2 inhibitor Phox-I2 also failed to protect against cytokines, but protected partially against high glucose + palmitate-induced cellular death. The highly selective Nox4 inhibitor GLX7013114 protected islet cells against both cytokines and high glucose + palmitate. However, as no osmotic control for high glucose was used, we cannot exclude the possibility that the high glucose effect was due to osmosis. It is concluded that Nox4 may participate in stress-induced islet cell death in human islets in vitro. We propose that Nox4 mediates pro-apoptotic effects in intact islets under stressful conditions and that selective Nox4-inhibition may be a therapeutic strategy in type 2 diabetes.

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Conflict of interest statement

XW, AE, NW, and P-OC have no conflict of interest to disclose. PW and EW have applied for the following patents: Wilcke M, Walum E, Wikström P. Thiophene-based compounds exhibiting nox4 inhibitory activity and use thereof in therapy. 2013 Patent application number PCT/EP2013/055218, and the European patent application No. 18171556.6 submitted 2018-05-09 protecting the Nox4 selective compound GLX7013114. The funder Glucox Biotech AB provided support in the form of salaries for authors EW and PW. Glucox Biotech AB has supported this study by generating, characterizing and providing NOX inhibitors. This does not alter our adherence to PLOS ONE policies on sharing data and materials. NOX inhibitors will be shared pending patent publication.

Figures

Fig 1
Fig 1
Immunoblot analysis of Nox2 and Nox4 in human islets (A-C) and EndoC-βH1 cells (D). Nox2 (A) and Nox4 (B) expression was assessed after 4h and 24h of IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt) and palmitate (1.5 mM in 2% BSA) + high glucose (20 mM) (PH) exposure. The expression of Nox2 and Nox4 was normalized to total ERK, which was used as a loading control. Results are means ± S.E.M for 5 independent experiments. * denotes P<0.05 using Student’s paired t-test. (C) Representative immunoblot image for Nox2, Nox4 and total ERK in human islet cells. (D) Representative immunoblot image for Nox2 (green), Nox4 (red), tubulin (green) and total ERK (red) in EndoC-βH1 cells.
Fig 2
Fig 2. Immunofluorescence analysis of Nox2 and Nox4 expression in human islet cells and EndoC-βH1 cells.
Islets and EndoC-βH1 cells were analyzed for Nox2/Nox4 immunofluorescence and counterstained with DAPI. Some cells were incubated for 24h with IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (cyt). Arrows point to EndoC-βH1 cells in late telophase with increased cytoplasmic Nox4 accumulation. Results are representative for 3 independent experiments.
Fig 3
Fig 3. ROS production in human islet cells.
(A) Quantification of ratio of DCF fluorescence to DAPI fluorescence intensity. Human islets were incubated at control condition (Ctrl) or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) for 24h. During the last 3h of the 24h incubation Nox inhibitors: DPI (0.2 μM), Dapsone (10 μM), GLX351322 (10 μM) and GLX481372 (6 μM) were added to the culture medium. each treatment group, 5 images (100–250 cells in each image) were taken. The intensities of green (DCF) and blue (DAPI) signals were quantified by Image J software. Results are means ± S.E.M for 4 donors. * denotes P<0.05 compared with PH using repeated measurements one-way ANOVA and Fisher LPD. For (B) Representative photographs showing DCF and DAPI fluorescence. M22 depicts GLX351322 and M72 depicts GLX481372.
Fig 4
Fig 4. Effects of DPI, GLX351322, GLX481372 and Dapsone on human islet cell viability and caspase 3 activation.
(A) Quantification of relative cell death (ratio PI/Bisbenzimide). Human islets were incubated at control conditions or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) for 24h with Nox inhibitors: DPI, Dapsone, GLX351322 and GLX481372 (same concentrations as described in Fig 3 and present during the entire 24h exposure period). For each treatment group, 5 images (100–250 cells in each image) were taken. Islets were photographed in an inverted fluorescence microscope and the intensities of red (PI) and blue (Bisbenzimide) signals were quantified using Image J software. Results are means ± S.E.M for 7 human islet donors. * denotes P<0.01 compared with PH using repeated measurements one-way ANOVA and Fisher PLD. (B) Representative images of cleaved caspase 3 staining (red) after a 24h palmitate + high glucose incubation with and without Nox inhibitors.
Fig 5
Fig 5. Effects of ML-171, Phos-I2 and GLX7013114 on human islet cell viability.
Human islets were incubated at control condition, with IL-1β (20 ng/ml) + IFN-γ (20 ng/ml) (A), or with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) (B) for 2 days with or without Nox1 inhibitor ML-171 (2 μM), Nox2 inhibitor Phos-I2 (2 μM) and Nox4 inhibitor GLX7013114 (1 μM). Islets were photographed in an inverted fluorescence microscope and the intensities of red (PI) and blue (Bisbenzimide) signals were quantified using Image J software. Results are means ± S.E.M for 7 human islet donors. * denotes P<0.05 compared with cyt or PH, respectively, using repeated measurements one-way ANOVA and Fishers PLD post-hoc test.
Fig 6
Fig 6. Effects of ML-171, Phos-I2 and GLX7013114 on EndoC-βH1 cell ROS production and viability.
(A) Flow cytometry quantification of DCF (total ROS) and MitoSOX (mitoROS) intensities at control conditions. Results are means ± S.E.M for 3 independent experiments. * denotes P<0.05 compared with control using Student’s paired t-test. (B) Flow cytometry quantification of cell death after a 2-day incubation with cytokines (cyt) supplemented with 0.2 μM, 0.5 μM or 1.0 μM of Nox1 inhibitor ML-171, Nox2 inhibitor Phos-I2 or Nox4 inhibitor GLX7013114, respectively. Results are means ± S.E.M for 7 independent experiments. (C) Flow cytometry quantification of cell death after 24h incubation with palmitate (1.5 mM + 2% BSA) + high glucose (20 mM) (PH) supplemented with 0.2 μM, 0.5 μM or 1.0 μM of Nox1 inhibitor ML-171, Nox2 inhibitor Phos-I2 or Nox4 inhibitor GLX7013114, respectively. Results are means ± S.E.M for 8 independent experiments.
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Grants and funding

The present study was supported in part by Olle Engkvist Byggmästare, the Swedish Research Council (2017-01343), the Swedish Diabetes Association, the Novo Nordisk Foundation, the family Ernfors Fund, the Swedish Child Diabetes Fund and EXODIAB. The funder Glucox Biotech AB provided support in the form of salaries for authors EW and PW. Glucox Biotech AB has supported this study by generating, characterizing and providing NOX inhibitors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.